Therapeutic treatments to the CNS necessarily require delivery of the requisite biological therapeutic to the target cells and organs to achieve physiologically significant expression levels without toxicity. Gene therapy has the potential, though largely still unrealized, to significantly advance clinical medicine. Long term expression after gene therapy is useful for diseases which require chronic levels of protein expression, such as inherited enzyme deficiencies. However, the risks and duration of gene delivery must also be closely matched to the proposed clinical application.
Gene therapy affecting the CNS has been the target of concentrated research efforts for many years. However, gene delivery and expression to the CNS has focused primarily on long term delivery methods using viral vectors. Nevertheless, while viral vector systems have been used to transfect cells in vitro and in vivo, they have not yet proven safe in a patient. For clinical applications in which only short-term gene expression is required or warranted, the delivery of nucleic acids by means of a non-viral cationic lipid may provide a more favorable risk/benefit analysis. Moreover, cationic lipid-mediated transfection advantageously offers low immunogenicity, ease of preparation, and the ability to transfect vectors of nearly unlimited size (Mountain, Trends in Biotechnol. 18:119-128 (2000)), as compared with the use of viral vectors. However, clinical trials using cationic lipids for delivery have primarily examined applications requiring brief expression of transgenes, such as therapies aimed at direct or immunological killing of tumor cells.
In proliferating cells, the majority of the DNA enters the nucleus through passive movement. This occurs during the nuclear membrane degradation stage of mitosis (Wilson et al., supra, 1995; Melchior et al., supra, 1998). However, the amount of DNA that is able to cross the nuclear envelope in non-proliferating cells by passive movement through the NPC has been assumed to be negligible (Aronsohn et al., supra, 1998), creating an additional barrier in the development of an efficient DNA delivery system (Bally et al., Adv. Drug Deliv. Rev. 38:291-315 (1999)); Girao da Cruz et al., Biochim. Biophys. Acta 1510:136-151 (2001)); Zabner et al., J. Biol. Chem. 270:18997-19007 (1995)). However, trials have not examined cationic lipid delivery methods of gene sequences in vivo for the rapid, transient expression of neuroprotective proteins in the central nervous system, or for attenuating cell death following CNS trauma.
The cerebral spinal fluid (CSF) is an effective way to express gene vectors in the brain. Rennels et al., Brain Res. 326:47-63 (1985)) proposed a rapid perivascular flux from CSF to the extracellular fluid to account for rapid fluid circulation throughout the CNS. Intra-ventricular injection in rats of an adenoviral vector for β-gal and IL-2 receptor antagonist (Betz et al., J. Cerebral Blood Flow and Metabolism 15:547-551 (1995); Yang et al., Brain Res. 751:181-188, (1997)) showed staining for f3-gal confined primarily to cells lining the ventricles. Despite this apparently limited expression, a reduction of stroke size in permanent focal ischemia was seen and the authors concluded that the ependyma does not form a barrier to movement of substances between CSF and brain.
Many investigators have demonstrated the use of viral vectors for transferring genes into various tissues, including into the CNS (Barr et al., Gene Ther. 1:51-58 (1994); Engelhardt et al., Human Gene Ther. 4:759-769 (1993); Hermens et al., J. Neurosci. Meth. 71:85-98 (1997); Shimohama et al., Mol. Brain Res. 5:271-278 (1989)). Leading viral vector systems include recombinant retrovirus (for replicating cells), adeno-(AV) and adeno-associated (AAV) virus, retroviral (RV), lentiviral, and herpes simplex virus vectors (HSV). For example, fetal myocytes (cardiac cells) that had been transfected with HSP70 using a viral vector were protected when subjected to simulated ischemia (Mestril et al., J. Mol. Cell Cardiol. 28:2351-2358 (1996)). Giffard et al. transfected astrocytes using a retroviral vector for HSP70 and reported protection of astrocytes and neurons from oxygen-glucose deprivation (Papadopoulos et al., Neuroreport 7:429-432 (1996)) in the same cell culture, even though only the astrocytes were transfected (Xu et al., Soc Neurosci Abstracts 23:845.1323:845.13 (Society for Neuroscience Annual Meeting, New Orleans, 1997)).
Viral vectors have also been used to protect the CNS by direct injection (Hermens et al., supra, 1997). Fink et al. used a herpes simplex vector to show improved neuron survival in a rat middle cerebral artery occlusion model (Yenari et al., Soc. Neurosci. Abstracts 23:547.12 (Society for Neuroscience Annual Meeting, New Orleans, 1997)), while the use of HSV to transfect non-dividing cells in the CNS has been reported by Naldini et al., Science 272:263-267 (1996)). Retroviral and HSV vectors (Geller et al., Proc. Natl. Acad. Sci. USA 87:1149-1153 (1990)) have been used in experimental trials for the treatment of human CNS tumors (Karpati et al., Trends in Neurosciences 19:49-54 (1996)), but none of these viral-based gene delivery methods teach how to safely deliver gene sequences for CNS neuroprotection without the risks of viral vectors.
Inherent risks associated with the use of a viral vector in a patient are known, and experimental use of such vectors has resulted in at least one well-publicized patient death secondary to liver failure after adenoviral gene therapy in 1999. Inflammation is a known complication resulting from the use of the common adenoviruses for viral gene therapy (Byrnes et al., Neuroscience 66:1015-1024 (1995); Kajiwara et al., Human Gene Ther. 8:253-265 (1997). An ineffective T-cell response in the brain may account for a prolonged response to adenoviral vectors in brain tissue (Byrnes et al., Gene Ther. 3:644-651 (1996)). An adenovirus expressing the herpes simplex thymidine kinase gene was tested in rats and primates in intracranial injections, but a dose dependent localized necrosis, mild gliosis, focal astrocytosis trace meningitis, and perivascular cuffing was described (Smith et al., Human Gene Ther. 8:943-954 (1997)). Undesirable persistence of defective HSV-1 vectors in rat brain has also been reported (Starr et al., Gene Ther. 3:615-623 (1996). Many other drawbacks, including production difficulties, have been reported. For example, in one issue alone of Molecular Therapy, the journal of the American Society for Gene Therapy, several articles reported that significant problems still remained with the use of viral vectors, including: oncogenesis following lentiviral fetal and neonatal murine delivery; inadvertent gene transfer to male germ-line cells following retroviral gene delivery; and encapsulation and in vivo persistence of prokaryotic sequences during the production of AAV vectors.
Moreover, although viral vectors can achieve transduction, producing extremely high levels of protein expression, such high levels of expression and infectivity may be more than is needed to be of physiologic significance in vivo, even if they could be routinely achieved. Viral vectors may, in fact, overwhelm the cellular protein producing apparatus of the cell in vivo (Peltekian et al., J. Neurosci. Meth. 71:77-84 (1997). Despite the recognized deficiencies associated with the use of viral vectors to deliver the gene sequences, the literature also fails to teach how to avoid significant inflammatory and/or immune responses. Indeed, there are no reports of non-viral, rapid, widespread CNS transfection without the requirement of craniotomy or burr holes in the skull, followed by injection into brain or lateral ventricle.
In an attempt to overcome the problems associated with viral delivery, researchers have proposed a number of non-viral methods for delivering polynucleotides into neuronal cells and tissues. Calcium precipitation and electroporation are of extremely limited usefulness in vivo in the CNS. Jiao et al., Biotechnology 11:497-502 (1993) bombarded fetal brain tissue with DNA that had been precipitated onto gold pellets (the Accell device) and demonstrated transfection of isolated neuron and glial cells. Biewenga et al., J. Neurosci. Meth. 71:67-75 (1997) reviewed the specific parameters of the use of biolistics (“gene gun”) to transfect neurons in culture. Naked DNA can be transfected into skin (Yu et al., J. Invest. Dermatol. 112:370-375 (1999)) and high ionic strength carriers, such as liposomes, can be used to augment the low baseline efficiency and durability of gene expression in this tissue setting (Chesnoy et al., Mol. Ther. 5:57-62 (2002); Alexander et al., Human Mol. Genet. 1995; 4: 2279-2285 (1995)). However, there are few reports in the literature of widespread expression in CNS using non-viral methods, particularly methods with the potential for clinical usefulness and none are non-invasive. See, e.g., Zhu et al., Gene Ther. 3:472-476 (1996), who reported use of cationic lipid mediated gene transfer of the herpes simplex virus thymidine kinase (tk) gene into glioma tumor cells in vivo in rats, using a continuous infusion pump. However, such non-viral methods involve direct injection into the brain, requiring a surgical procedure (brain surgery) prior to injection
Cationic lipid-mediated gene transfer is particularly suited for transient gene expression, both in basic research and in selected clinical applications. Cationic lipids are used to protect plasmid DNA from DNAse I digestion, from endogenous DNAses in the extracellular environment and CSF (Luo et al., Nature Biotechnology 18:33-37 (2000)). Hundreds of clinical gene therapy trials have been completed or remain currently in progress (updated at J. Gene Medicine Web site at http://www.wiley.co.uk/genetherapy), mainly for the treatment of melanoma, head and neck, and ovarian cancer. At least 22% of the reported human clinical protocols are either non-viral lipid-mediated, or utilize non-viral DNA delivery. Thus, the safety of non-viral mediated gene transfer is no longer an issue, and cationic lipid-mediated gene delivery avoids many of the potential objections to the use of viral DNA vectors. Moreover, the results normally reflect a dramatic improvement over therapies using naked DNA delivery.
Cationic lipids commonly have a polar head group and non-polar symmetric or dissymmetric carbon based (fatty acid) tail, which gives membrane fluidity to the lipoplex. Negatively charged nucleic acids condense and self-assemble into heterogeneous complexes of lipids and nucleic acids when mixed with cationic lipids (Feigner et al., Annals NY Acad. Sci. 772:1126-1139 (1995)). The structure and size of these complexes affect transfection efficacy and vary with temperature, concentration, charge ratio, buffer, time, and lipid composition. Numerous laboratories (e.g., Feigner et al., Proc. Nat'l. Acad. Sci. USA 84:7413-7437 (1987); Byk et al., Drug Develop. Res. 50:566-572 (2000); Niedzinski et al., Mol. Ther. 6: 279-286, 2002)) have investigated the limiting parameters of cationic lipid-mediated transfection with the goal of improving transfection efficiency.
While cationic lipid-mediated delivery is useful for delivery of nucleic acids, either mRNA or DNA, see Feigner et al., supra, 1987), transfection of primary cell lines (including for neuronal and glial cells of the CNS) remains a problem (Wangerek et al., J. Gene Med. 3:72-81 (2001)). There are four general barriers to lipid-mediated DNA transfection: 1) transport of the nucleic acid/lipid complex in the extracellular environment; 2) association and uptake of the nucleic acid/lipid complex by the target cell (Bally et al., supra, 1999); Feigner et al., supra, 1995); 3) intracellular DNA release from the nucleic acid/lipid complex (Girao da Cruz et al., supra, 2001); and 4) translocation of DNA to the nucleus (Mortimer et al., Gene Therapy 6:403-411 (1999)). The primary barrier to DNA transfections in post mitotic cells is assumed to be DNA translocation to the nucleus (Zabner et al., supra, 1995).
Neuronal cells are regarded as particularly difficult to transfect with non-viral techniques, although adenovirus or HSV have met with some success in the CNS (Krisky et al., Gene Therapy 5:1593-1603 (1998)). This difficulty is generally attributed to markedly reduced or absent mitotic activity (Wangerek et al., supra, 2001). In proliferating cells, nuclear translocation is mainly passive, occurring during mitosis as the nuclear membrane breaks-down (Bally et al, supra, 1999; Wilke et al., Gene Therapy 3:1133-1342 (1996); Nicolau et al., Biochim. Biophys. Acta 721:185-190 (1982)). Some nuclear translocation also occurs in non-proliferating cells, probably as a result of passive movement through the nuclear pore complex (NPC) (Mattaj et al., Ann. Rev. Biochem. 67:265-306 (1998); Wilson et al., J. Biol. Chem. 270:22025-22032 (1995)). To improve efficiency of lipid-mediated DNA transfer some investigators have used nuclear localization sequences (NLS) (Aronsohn et al., J. Drug Targeting 5:163-169 (1998); Melchior et al., Trends Cell. Biol. 8:175-179 (1998)) to target the non-proliferating cells, and thus to facilitate DNA entry into the nucleus.
Methods for otherwise avoiding the necessity of nuclear translocation of DNA have also been reported, such as delivery of T7 promoter DNA plasmid systems to T7 polymerase expressing cells (Brisson et al., Human Gene Therapy 10:2601-2613 (1999)). However, the T7 system is not useful in most basic research or clinical applications. Lipid-mediated RNA delivery to proliferating cells (Malone et al., Proc. Nat'l. Acad. Sci. USA 86:6077-6081 (1989)), as well as intramuscular injection of naked RNA has been previously described (Wolff et al., Science 247:1465-1468 (1990)). Jirikowski et al., Science 255:996-998 (1992)) reported the uptake of mRNA in vivo by neurons, following direct injection in the rat hypothalamus to correct diabetes insipidus. Kariko et al., J. Neurosci. Meth. 105:77-86 (2001)) demonstrated local expression by in situ and immunocytochemical techniques after injection of RNA complexed with lipofectin (Gibco/BRL) into brain parenchyma. There is also evidence that, at least, some mRNAs may be actively taken up by neural tissue and transported throughout the CNS (Mohr et al., EMBO J. 10:2419-2424 (1991)). However, none of these reports describe methods for rapid and widespread, rapid gene expression in the CNS after delivery of mRNA vectors into the CNS.
The leading non-viral gene therapy method involves the use of mRNA or DNA as a lipid/nucleic acid complex (“lipoplex”), with or without membrane proteins to provide targeting specificity. See, U.S. Pat. Nos. 5,869,715; 5,925,623; 5,824,812, (each by Nantz et al.), and related publications. Hecker et al., Molecular Therapy 3:375-384 (2001) described DNA expression using lipoplexes, and the methods described in the 2001 were used by Anderson et al., Human Gene Therapy 14(3):191-202 (2003) to examine the functional integrity and protection from degradation of mRNA/cationic complexes (lipoplexes) for more than 4 hours in human cerebrospinal fluid (hCSF) in vitro (as compared with rapid <5 min disappearance of non-lipid-complexed mRNA), and preliminary findings regarding expression of these lipoplexes in vivo (in vitro transcribed mRNA vectors encoding Hsp70 and luciferase were delivered to the lateral ventricle of brains of rat models). Expression was noted in coronal sections throughout the rat brain, confirming the potential for lipid-mediated mRNA delivery to the CNS. However, these publications demonstrated results only in the rat brain, and only following direct injection into the lateral ventricle of the rat brain. No evidence was reported indicating success in a non-human primate model or in higher order intact animals, or suggesting the possibility that intrathecal delivery to the CSF would be effective in humans.
A common problem associated with non-viral nucleic acid delivery techniques is that the amount of exogenous protein expression produced relative to the amount of exogenous nucleic acid administered remains too low for most diagnostic or therapeutic procedures. Low levels of protein expression are often a result of a low rate of transfection of the nucleic acid or the instability of the nucleic acid. As a result, despite numerous research efforts directed at finding efficient methods for nucleic acid delivery, most known techniques have failed to provide sufficient cell transfection to achieve the desired protein expression to be of clinical value. While prior art publications have shown rapid uptake, distribution, and expression of exogenous DNA and mRNA in the rat brain using GFP and luciferase reporter gene sequences, the current literature offers no non-invasive method for achieving rapid and extensive gene expression in the brains of human patients or primate models via cerebrospinal fluid administration free from the inherent risks and difficulties associated with the use of viral vectors. For example, although Schwartz et al., Gene Therapy 3:405-411 (1996) described modest expression of the reporter gene enzymes luciferase and β-galactosidase in rat brain after direct injection of up to 150 μg of DNA plasmid, they reported no additional efficacy using several standard cationic lipids, and their peak in enzyme assay signal was not reached until 48 hours post-administration. Primates have more robust and versatile immunological responses.
Heat shock proteins (HSP) are members of a highly conserved family of molecular chaperones that play important roles in normal cellular function and survival. They act as molecular chaperones expressed constitutively and rapidly induced in response to various types of stress, including heat shock, ischemia, oxidative stress, glucose deprivation, and exposure to toxins and heavy metals (Kiang et al., Pharmacol. Ther. 80:183-201 (1998)). The Hsp70 gene encodes a 44 kDa amino terminal ATPase domain, and a carboxyl terminal domain that contains the 18 kDa peptide or substrate binding domain followed by a 10 kDa stretch terminating in the highly conserved EEVD sequence (O'Brien et al., J. Biol. Chem. 271:15874-15878 (1996); Ohno et al., FEBS Lett. 576:381-386 (2004); Rajapandi et al., Biochem. 37:7244-7250 (1998); Wang et al., J. Biol. Chem. 268:26049-26051 (1993)).
Whole animals, isolated organs and cells subjected to heat shock are protected against a subsequent near lethal ischemic or hypoxic event. For example, Fink et al. transfected hippocampal neurons with the Hsp70 gene using a HSV vector to show improved neuron survival in a rat MCA occlusion model, and demonstrated what the authors described as “the first published report of protection following heat shock protein transfection in CNS neurons” (Fink et al., J. Neurochemistry 68:961-969 (1997)).
More recently, studies have shown that Hsp70 overexpression protects cells from death induced by various insults that cause either necrosis or apoptosis, including near lethal hypoxia and ischemia/reperfusion, by inhibiting multiple cell death pathways (Giffard et al., J. Neurosurg. Anesthesiol. 16:53-61 (2004); Steel et al., J. Biol. Chem. 279:51490-51499 (2004)). The induction of protective intracellular responses (“endogenous”) by heat shock is not clinically useful, but the enforced overexpression with viral vectors, as a transgene, or pharmacological induction of Hsp70, all decrease injury after cerebral ischemia and protect both neurons and glia (Giffard et al., J. Exp. Biol. 207:3213-3220 (2004); Hoehn et al., J. Cerebral Blood Flow Metab. 21:1303-1309 (2001); Lu et al., J. Neurochem. 81:355-364 (2002); Rajdev et al., Ann. Neurol. 47:782-791 (2000)). Nevertheless, the ability of Hsp70 to provide neuroprotection has only been demonstrated in limited viral models on rodent species, but, to date, it has not been reported in non-human primates or in humans.
It has been previously reported that astrocytes expressing elevated levels of inducible Hsp70 are protected from oxygen-glucose deprivation (OGD), hydrogen peroxide (H2O2) exposure, and hyperthermic insult (Papadopoulos et al., Neuroreport 7:429-432 (1996); Xu et al., Biochemistry 35:5616-5623 (1996); Xu et al., Neurosci Lett. 224:9-12 (1997)). However, it was not clear whether the protective effect of Hsp70 was caused by direct interaction with misfolded protein or, whether it resulted from the anti-apoptotic and anti-necrotic effects of Hsp70. Sun et al., J. Cerebral Blood Flow and Metabolism, 26(7):937-950 (July 2006) (Epublished November 2005), using two mutants of Hsp70, confirmed the importance of protein folding to ischemic protection, and showed that the peptide binding domain of Hsp70 was sufficient for protecting cells from ischemia in vitro (primary astrocyte culture) and in vivo (reduced infarct size and focal ischemic injury as induced by transient middle cerebral artery occlusion, and improve neurological function). These results are consistent with prior reports showing that a deletion mutant, containing the peptide binding domain of Hsp70, but lacking the ATPase domain, is still capable of protecting cells from heat (Li et al., J. Biol. Chem. 275:25665-25671 (1992)), serum withdrawal (Ravagnan et al., supra, 2001) and heat-stress induced apoptosis (Volloch et al., FEBS Lett. 461:73-76 (1999)). Thus, the significant protective effect of the mutants suggest that peptides, such as Hsp70, specifically the carboxyl-terminal domain of Hsp70, could offer a useful therapeutic strategy for the treatment of stroke and neurodegenerative diseases if safely delivered to the target CNS.
In addition, there is strong evidence that Hsp70 can protect cells from toxicity due to misfolded, aggregated proteins associated with neurodegenerative diseases (Dong et al. Molecular Therapy 11:80-88 (2005); Muchowski et al., Nat. Rev. Neurosci. 6:11-22 (2005); Tidwell et al., Cell Stress Chaperones 9:88-98 (2004)). For example, overexpression of Hsp70 suppressed degeneration and improves motor function in a transgenic mouse model of SCA1 (Cummings et al., Hum. Mol. Genet. 10:1511-1518 (2001)). Similarly, the overexpression of Hsp70 reduces the toxicity of mutant α-synuclein in Parkinson's disease (Auluck et al., Science 295:865-868 (2002)).
It is evident from the prior art that HSP70 offers great potential as a neuroprotectant before injury, and as a therapeutic solution immediately after CNS or spinal cord injury, if it can be safely delivered to the site and cells where it is needed. However, the reported induction of protective intracellular responses by heat shock proteins is not clinically useful because delivery via a viral vector carries the inherent risks to the patient, and because rapid and widespread distribution that is required for use of the protective HSP gene sequences has not been provided. Sun et al. supra, 2005 provide valuable insight into possible, albeit unproven, mechanisms by which HSP70 may be effective, but no prior art publication has taught how to integrate all of the necessary knowledge, including formulation, synthesis, packaging, delivery, efficacy, safety and methodology to successfully administer therapeutic or preventative compounds to the human CNS. Thus, until the present invention, a need has remained in the art for non-viral, lipid mediated methods, that do not involve surgical intervention needed for direct injection to the brain, for a safe, short-term delivery of nucleic acids, particularly mRNA, encoding therapeutic proteins, e.g., HSP, to cells of the CNS, and for the rapid expression and widespread distribution of the therapeutic proteins for in vitro or in vivo applications, including clinical use. Such methods could promote, for example, nervous system cell repair and regeneration in vivo, and/or prevent or decrease the severity of ischemic damage due to, e.g., spinal and/or brain injury, including damage during surgery.